The genus mycobacterium is responsible for more suffering worldwide than all other bacterial genera combined. Mycobacteria are classified into two broad categories. The first category, the M. tuberculosis complex, includes M. tuberculosis, M. bovis, M. microtti, and M. africanus. The second category includes all other species and is referred to as nontuberculosis mycobacteria (NTM) or mycobacteria other than tubercule bacilli (MOTT) which includes, among others, M. kansasii, M. marinum, M. similae, M. scrofulaceum, M. szulgai, M. gordonae, M. avium, M. intracellulare, M. ulcerans, M. fortuitum, M. chelonae, M. xenopi, and M. malmoense. Currently, over 60 species of mycobacteria have been defined. Of all the culturable mycobacteria, only M. tuberculosis is an obligate pathogen.
Tuberculosis (TB) which is caused by infection with M. tuberculosis or M. bovis remains one of the most significant diseases of man and animals (1-3) and continues to inflict a huge cost on society with regard to human and animal health and financial resources (4-7). The only vaccine currently available for the prevention of TB is a live attenuated vaccine, Bacille Calmette-Guerin (BCG), derived from Mycobacterium bovis. BCG possesses many of the qualities of an ideal vaccine: it is cheap to produce and administer, it is safe and has been shown to be efficacious in many circumstances, especially against severe and fatal tuberculosis in children (8). However, BCG has been found to give variable efficacy in a number of clinical trials. In the Medical Research Council trial in the United Kingdom, BCG imparted 77% protection (9) while, at the other end of the spectrum, in the largest clinical trial in India it exhibited zero protective efficacy (10). Although BCG generally gives poor protection against pulmonary tuberculosis in adults, it remains the “gold-standard” against which candidate TB vaccines of improved efficacy are measured. With the advent of the HIV/AIDS pandemic, concern has been raised over the safety of BCG. Since BCG can be pathogenic in situations of compromised or deficient immunity (11), vaccination with BCG can be contraindicated for those very individuals most at risk of contracting tuberculosis.
Problems surrounding the lack of universal efficacy and safety of BCG have resulted in increased efforts to develop a new generation of tuberculosis vaccines. One approach being pursued is the generation of subunit vaccines requiring inoculation of mycobacterial nucleic acid, protein(s) or peptides in adjuvant. While individual proteins generally have only marginal efficacy, protein mixes work much better (12), suggesting immunity to numerous antigens is required for full protection. This is supported by the observation that DNA vaccination with multiple antigens has an additive effect on protective efficacy (13). Another approach has used molecular genetic tools to generate mutant members of the TB complex that are attenuated and avirulent to an immunocompromised host (14). For example, deletion of genes involved in amino acid and purine biosynthesis resulted in auxotrophic mutants of BCG that were unable to persist in both immunocompetent (15, 16) and severely immunocompromised mice (17). However, the mutants were able to persist long enough to express metabolic antigens and engender a degree of protective immunity. It has been suggested that such mutants could be used to vaccinate individuals at risk of developing compromised or deficient immunity (17), although concerns remain regarding the use of genetically modified live vaccines in either humans or livestock (14, 18).
Vaccines based on killed whole cell preparations of mycobacteria have classically conferred little to no specific protection to subsequent challenge with virulent mycobacteria (19-22), presumably because the important protective antigens are only expressed when the bacteria are metabolically active (21, 23, 24). There are exceptions to this (25, 26) and it has been proposed that in terms of generating protective immunity, the particular antigens that are presented may be less important than the way in which they are presented (27, 28). The majority of vaccination studies with killed preparations of mycobacteria have used heat as the method of killing. However, this treatment may significantly denature important antigens and could account for the disappointing results generally seen with such vaccines (29). An alternative to heat inactivation is treatment with formalin. Formalin inactivation was first used in the 1920's to detoxify the diphtheria toxin isolated from cultures of Corynebacterium diphtheriae (30, 31); the approach still used for the production of the vaccine (32). Formalin treatment of whole mycobacteria has the advantage of killing the organism while retaining the antigenic integrity of many of the proteins present (33).